Characterizing cell viability and other cell features can provide useful information with respect to a wide range of applications. However, methods presently employed are quite complex and time consuming. The cell cycle represents the most fundamental and important process in eukaryotic cells. Being an ordered set of events, culminating in cell growth and division into two daughter cells, the cell cycle is tightly regulated by defined temporal and spatial expression, localization and destruction of several cell cycle regulators. Cyclins and cyclin-dependent kinases (CDK) are major control switches for the cell cycle, causing the cell to move from G1 to S or G2 to M phases. In a given population, cells will be distributed among three major phases of cell cycle: G1/G0 phase (one set of paired chromosomes per cell), S phase (DNA synthesis with variable amount of DNA), and G/M phase (two sets of paired 2 chromosomes per cell, prior to cell division).
Because cell cycle dysregulation is such a common occurrence in neoplasia, the opportunity to discover new targets for anticancer agents and improved therapeutics has been the focus of intense interest. The cell cycle analysis has applicability to a variety of areas of life science research and drug development, including cancer biology, apoptosis analysis, drug screening and measuring health status of cell cultures, e.g. in bioreactors.
The most common approach to determine the cell cycle stage is based on measurement of cellular DNA content. DNA content can be determined using fluorescent, DNA-selective stains that exhibit emission signals proportional to DNA mass. Cellular fluorescence is measured by flow, image or laser scanning cytometry. A variety of fluorochromes can be used for staining of DNA.
DNA staining is typically performed on cells permeabilized with either non-ionic detergents or alcohol fixation. A plethora of protocols based on these two approaches for cell permeabilization have been published in the literature. Although most of these protocols are relatively simple and applicable for many cell types they have several drawbacks and limitations. Firstly, the prior art methods require that cells are in suspension and, accordingly, adherent cells lines have to be detached prior to analysis. Secondly, they contain washing steps, calling for centrifugation that often results in cell loss. Thirdly, they promote cell aggregation, which hampers DNA content quantification of individually cells.
The method described herein is a simple method for quantification DNA and/or RNA of individually cells by releasing nuclei from the cells and performing nucleic acid quantification on these nuclei.
A widely used method to monitor cell concentration of adherent cells is to count released nuclei. Release of nuclei is often achieved using solutions containing mild detergents, such as Triton-X100. It has been described that Triton X-100 in combination with citric acid efficiently releases nuclei from anchorage-dependent cultures and cultures containing cell aggregates (Lin et al., A rapid method for counting cell nuclei using a particle sizer/counter (1991). Biotechnology Techniques, 5, 153-156). Lin et al used a solution of 100 mM citric acid, 1% triton X-100 and 0.1 mg/ml crystal violet. This solution has a pH value below 2, and a non-fluorescent dye. The method of Lin et al is used for cell counting and not for determining the amount of nucleic acid within the nuclei.
When quantifying nucleic acid of nuclei to assess stages of cell cycle, it is important to obtain results without to much “noise”. The invention described herein makes it possible to obtain good results from labelled nucleic acid at low magnification and with a low degree of noise.